STIM/Orai-mediated store operated Ca2+ entry may contribute to overproduction of TSLP in epidermal cells isolated from atopic dermatitis mice

[Speaker] Shuhei Kobayashi:1
[Co-author] Rieko Matsui:1, Yumeka Yamada:1, Hiroaki Kito:1,2, Susumu Ohya:1,2, Masanori Fujii:1
1:Department of Pharmacology, Kyoto Pharmaceutical University, Japan, 2:Department of Pharmacology, Graduate School of Medical Sciences, Nagoya City University, Japan

[Background] Atopic dermatitis (AD) is a highly pruritic, chronic inflammatory skin disease. Thymic stromal lymphopoietin (TSLP) is highly expressed in the epidermis of AD patients. Also, TSLP is known to induce Th2 immune responses and itch; however, the mechanism of how TSLP is produced in AD conditions is not fully understood. We have reported a unique, diet-induced AD mouse model (Fujii et al., Exp. Dermatol., 14, 460-468, 2005). In the present study, we examined the TSLP production capability of epidermal cells isolated from the AD model mice and determined its mechanism of action.
[Methods] HR-1 hairless mice were fed a normal or the special (called HR-AD) diet. Epidermal sheet was obtained by treatment with dispase overnight at 4 °C, and then was treated with trypsin at 37 °C to isolate epidermal cells. The isolated epidermal cells were incubated in the normal medium for 24 h at 37 °C. At the indicated time point in cultivation, mRNA and intracellular and extracellular protein of TSLP were determined by real time PCR and ELISA, respectively.
[Results] TSLP mRNA and intracellular and extracellular TSLP protein were significantly increased with a peak at 3 h, 6 h, and 24 h, respectively, after the cultivation in epidermal cells isolated from special diet-fed AD mice, whereas such TSLP increase was not observed in normal mice. The TSLP overproduction was almost completely inhibited by the Ca2+ chelator ethylene glycol tetraacetic acid, suggesting a requirement of extracellular Ca2+. Treatment with transient receptor potential channel inhibitors, such as Gd3+, ruthenium red and La3+, did not affect the TSLP overproduction. On the other hand, the TSLP overproduction was partly but significantly suppressed by 2-aminoethoxydiphenylborate, which blocks IP3 receptors. Furthermore, the TSLP overproduction was almost completely inhibited by treatment with either ML-9, which inhibits plasma membrane interaction of stromal interaction molecule 1 (STIM1), preventing store-operated Ca2+ entry (SOCE), or BTP-2, which blocks the Ca2+ release-activated Ca2+ channel Orai-1.
[Conclusions] Epidermal cells isolated from AD mice can produce high amount of TSLP, perhaps by STIM/Orai-mediated SOCE.
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